Impact of mercury on photosynthetic performance of Lemna minor: a chlorophyll fluorescence analysis

The purpose of this study was to evaluate the effectiveness of chlorophyll fluorescence analysis in detecting the effects of mercury (Hg) treatment in duckweed species Lemna minor. The results showed that Hg treatment (ranging from 0.0 to 0.4 µM) significantly impacted the plant's photosynthetic ability, with a decrease in variable chlorophyll fluorescence, energy fluxes, density of reaction centers, and performance index. Complete inhibition of electron transport was observed in plants treated with high Hg concentrations, and the quantum yield of primary photochemistry and the ratio of dissipated energy to absorption both decreased with increasing Hg concentrations. Performance Index (PI) was significantly affected by the Hg concentrations, reaching zero in plants treated with the highest Hg concentration. Overall, JIP analysis was found to be an effective tool for detecting deleterious effects of Hg in plants.

www.nature.com/scientificreports/ Lemna minor, also known as common duckweed, is widely distributed in a range of regions, from tropical to temperate and from freshwater to brackish waters 17 . The plant is native to Africa, Asia, Europe, and North America, but it also grows well in Australia and South America 18 . Researchers have found that L. minor has the ability to absorb high concentrations of HMs [19][20][21][22] . It has become a model system for ecotoxicological bioassays, genetic transformation, and industrial applications 23 . In addition to its applications in phytoremediation, L. minor has also been used to further our understanding of photoperiodic control of flowering 24 and photosynthesis research 25 .
The objective of this study was to examine the effectiveness of using Chl fluorescence analysis to detect mercury-induced stress in plants, using the aquatic angiosperm L. minor as a test subject. Additionally, exploring the potential of L. minor as a biomonitoring tool for Hg contamination in aquatic environments could provide valuable insights into the extent and effects of Hg pollution.

Results
Biophysical studies of Hg tolerance in L. minor. The  , which indicate the performance of active PSII reactions, underwent significant changes with increasing Hg concentrations in L. minor. At low Hg concentrations, ABS/RC did not change, but increased continuously with increasing Hg concentration in the media ( Fig. 2A). The highest values of ABS/RC were observed in plants exposed to 0.4 µM Hg. Similarly, TR/RC increased continuously with increasing the concentration of Hg in L. minor, with the highest values recorded when the plants were exposed to 0.3 µM Hg for 48 h (Fig. 2B). The electron transport flux per reaction center (ET/RC) showed a slight decrease with increasing Hg treatment up to 0.3 µM, followed by a slight increase at higher concentrations in L. minor (Fig. 2C). Mild to moderate Hg treatment had a limited effect on the dissipated energy flux per reaction center (DI/RC). The results showed that DI/RC increased gradually at lower concentration of Hg treatment (0-0.2 µM) and then steadily increased until the higher concentration (0.4 µM) (Fig. 2D).  (Fig. 4). The quantum yield of electron transfer, represented by ET/ABS (φEo), remained relatively unchanged during mild Hg treatment, but significantly declined about 3.5 times with increasing concentration of Hg. (Fig. 4).

Performance index.
The results of the study show that Hg had a pronounced impact on all photosynthetic parameters, including specific energy fluxes, phenomenological energy fluxes, density of reaction centers, and performance indexes, in L. minor exposed to various Hg concentrations. The performance index PI ABS was significantly affected by the exposure of Hg, with values reaching zero in the fronds of L. minor at high concentrations of 0.2-0.4 µM (Fig. 4). The lowest value of PI ABS was observed in plants subjected to 0.4 µM Hg. The overall effects of Hg-induced stress on all photosynthetic parameters are graphically depicted in the form of a radar plot (Fig. 4).

Discussion
The JIP test, which utilizes Chl a fluorescence analysis, provides a comprehensive assessment of photosynthetic parameters. This test allows for the evaluation of specific energy fluxes per Q A reducing PSII reaction center (ABS/RC, TR/RC, ET/RC, and DI/RC) and phenomenological energy fluxes per excited cross section (ABS/ CS, TR/CS, ET/CS, and DI/CS) [26][27][28] . The correlation matrix representing the performance of Chl fluorescence www.nature.com/scientificreports/ parameters was visualized using a heatmap. This heatmap provides a graphical representation of the interrelationships and correlations among the different fluorescence parameters (Fig. 5). Additionally, it enables the determination of the density of active and inactive PSII reaction centers (RC/CS) and various yield or flux ratios, such as φPo and φEo 29,30 . The test also provides an assessment of photosynthetic performance through the calculation of PI ABS 16 . The results of the Chl a fluorescence analysis in L. minor revealed a wide variation in response to different concentrations of Hg. As the intensity of Hg-induced stress increased, there was a continual decrease in the fluorescence signal (F M ) which suggests the denaturation of the light-harvesting complex (LHC) 26 . The changes observed in the Fo level are linked to the physical interaction of LHC II with the PS II antenna complex, which plays a role in regulating the transfer of energy from the PS II reaction center to other electron acceptor molecules. The decrease in F O and F M values suggests that the treatment with Hg resulted in the degradation of the PS II reaction center 31 .
The presence of a "K" peak in Chl fluorescence transients is widely recognized as an indicator of various types of stress in plants. According to 32 , this peak appears between the O and J peaks, forming the O-K-J-I-P pattern in Chl fluorescence transients and has been observed in various plants subjected to stress conditions such as heat stress [33][34][35] and drought stress 36 . However, in a recent study by the authors, it was found that the duckweed species did not exhibit the presence of a "K peak" in their Chl fluorescence curves. Several reports have suggested that the "K step" is related to the inactivation of the OEC 34,36-38 . The absence of a "K peak" in the Chl fluorescence curves of the duckweed species in the present study indicates that the OEC is not negatively impacted by HM treatment.
In conclusion, the absence of a "K peak" in the Chl fluorescence curves of the duckweed species studied suggests that the oxygen-evolving complex is not negatively impacted by HM treatment.
The results of the Chl a fluorescence analysis indicate that Hg in high concentrations plays a crucial role in disrupting the electron transfer from PSII to PSI. The analysis showed that increased Hg concentration reduced the performance index of PSII (PI ABS ) and the maximum quantum yield (F V /F M ). The disturbance in electron transfer, caused by Hg treatment, was evident in the JI and IP steps of the OJIP transient curves with increasing Hg concentrations. The results suggest that Hg interferes with light-dependent photosynthesis processes by reducing ATP synthase activity and restricting the flow of protons from the thylakoid lumen to the chloroplast stroma, leading to lumen acidification and a reduction in the oxidation of plastoquinone 39 . Additionally, Hg impacts the photosynthetic apparatus by decreasing the number of active reaction centers (RC) and reducing the rate of electron transfer 40 . The reduction of the rate of the terminal electron acceptors in PSI, as indicated by the I-step of the OJIP transient 41 , was also found to be reduced by Hg treatment.
The growth of L. minor with Hg resulted in a significant change in phenomenological energy fluxes per excited cross section compared to control plants. This change is attributed to the inhibition of various plant functions caused by Hg, which forms covalent bonds with the side groups of organic compounds such as proteins, leading to their inactivity 42 . The interaction of Hg with the SH-groups of proteins 43 is particularly significant and results in the inhibition of enzymes such as protochlorophyllide reductase and plastocyanin, as well as the Calvin cycle The impact of Hg toxicity on the PSII energy fluxes of L. minor was analyzed using energy pipeline leaf models and Biolyzer software. The model depicted the changes in the active and inactive PSII reaction centers per crosssection, as well as the flux of dissipated excitation energy at time zero (DI/CS). The ABS/CS, TR/CS, and ET/CS ratios, which represent the efficiency of light absorption, trapping, and electron transport of PSII, respectively, showed a decline with increasing Hg concentrations 47 . The decrease in ABS/CS indicated a reduction in energy absorbed per excited cross-section, while the decrease in ET/CS indicated lower energy absorption by antenna pigments and inactivation of reaction center complexes 48 . The gradual decrease in the TR/CS ratio demonstrated the significant impact of Hg on the trapping of reaction centers, which could be due to altered absorbance. The observations showed that Hg had a negative effect on PSII by decreasing the density of active reaction centers and increasing the density of inactive centers.
The F V /F M ratio is a measure of how effectively primary light energy is converted and captured, and it's commonly used to assess the quantum yield of primary photochemistry in PSII. If the F V /F M ratio declines, it suggests that the presence of Hg is reducing the quantum efficiency of PSII photochemistry either by slowing down the rate of primary charge separation or by disconnecting some minor antennae from PSII [49][50][51][52] . The area under . The inhibition of the J, I, and P phases, which correspond to various stages of electron transport in the photosynthetic process, can occur due to two reasons: first, inhibition of electron transport at the donor side of PSII, leading to the accumulation of P680 + , and second, due to a decrease in the pool size of the electron acceptor Q A −53,54 , which is also reflected by a decrease in the area parameter.
The inhibition of the photosynthetic apparatus by Hg is reflected in the decrease of the ratio of RC/ABS, which represents the density of active PSII reaction centers per Chl 55 . Studies have shown that Hg treatment results in a decrease in the number of active reaction centers per Chl, indicating maximum damage to the water splitting complex of PSII at a concentration of 0.4 μM 56 . This decrease also highlights the disruption of PSII photochemistry 56,57 .
The Performance Index on Absorbance basis (PI ABS ) is a widely used index for evaluating the primary photochemical reactions of PSII. It incorporates three key structural and functional characteristics of PSII, including the density of active PSII reaction centers per Chl (RC/ABS), the efficiency of light reactions (φ P O /(1−φ P O )), and the efficiency of dark redox reactions (ψ O /(1−ψ O )). This index is derived based on the Nernst equation for redox reactions 58 . The results showed a decline in the PI ABS with increasing Hg concentrations (0.1, 0.2, 0.3 and 0.4 μM) as presented in Fig. 4. This decline indicated a negative impact of Hg on the primary photochemical reactions of PSII 57 .
The changes in specific activity parameters of the PSII reaction center are crucial indicators of the absorption and utilization of light energy as well as the reaction center activity 59 . The PSII reaction center is designed to capture light energy for the subsequent transfer of energy. Any remaining energy is dissipated as heat 60 . Our www.nature.com/scientificreports/ results showed that the ABS/RC and TRo/RC increased significantly in leaves exposed to higher Hg concentrations. This is due to the reduction in the number of active reaction centers per unit area caused by Hg-induced stress, which increases the functional efficiency of the remaining active reaction centers and enhances the specific activity parameters per unit reaction center. This phenomenon is supported by the decrease in the RC/CS values. Furthermore, the increase in the DI/RC value at a Hg concentration of 0.4 μM suggests that the plant activates a self-protective mechanism to reduce excess energy in the PSII reaction center and increase the energy for heat dissipation per unit reaction center. Based on the present research, it is evident that Hg exposure can induce significant changes in photosynthetic processes in plants, causing inhibition in the photochemical reactions and damaging the PSII reaction centers. This information has important implications for future research on the impacts of HM toxicity on plant growth and productivity. Further studies are needed to understand the underlying mechanisms of Hg toxicity on photosynthesis and to develop effective strategies for mitigating the negative effects of Hg on plant growth and development. Additionally, this information can be useful for environmental monitoring programs and for risk assessment of HM pollution in agricultural and natural ecosystems. By better understanding the impacts of Hg on photosynthesis, it is possible to develop effective measures to protect the health of plants and ensure the sustainability of food production systems.

Measurement of polyphasic chlorophyll fluorescence kinetic. In the experiment, measurements
were performed on dark-adapted Lemna fronds using the Handy PEA instrument (by Hansatech Instruments Ltd., located in Norfolk, UK) after a minimum of 30 min of dark adaptation. Fluorescence rise OJIP curves were induced through a 1-s pulse of red light (650 nm, 3500 μmol/m 2 s). The fluorescence transients were recorded over a leaf area of 4 mm diameter using a red-light source with a peak at 650 nm and an intensity of 3000 µmol/ m 2 s, which was sufficient to close all PSII reaction centers and obtain a true F M fluorescence intensity. This was achieved through the use of a high-intensity LED array consisting of three light-emitting diodes. A measuring time of one second was consistently utilized throughout the experiment. Following primary fluorescence data were obtained from OJIP test:

Conclusion
The present investigation demonstrated that the increasing concentration of Hg had a negative impact on L. minor. Chlorophyll fluorescence analyses suggested that the photosynthesis process was primarily affected under Hg treatments. Hg treatment may hinder the functionality of PS II and ATP synthesis, which ultimately led to negative effects on duckweed survival. Hg exposure affected phenomenological energy fluxes per excited crosssection and specific activity parameters of the PSII reaction center. Energy absorption, trapping efficiency, and energy dissipation were all affected, which can be attributed to Hg's interaction with proteins and inhibition of key enzymes involved in photosynthesis. Furthermore, high concentrations of Hg disrupted electron transfer from PSII to PSI, resulting in reduced PI ABS and F V /F M of PSII. The OJIP parameters analyzed in this paper have the potential to serve as an effective tool for quickly identifying the main way in which harmful substances affect the photosynthesis in plants.

Data availability
The data and materials that support the findings of this study are available from the corresponding author upon request.